Promoters and utilization thereof

ABSTRACT

The present invention relates to improved promoters and utilization thereof, in particular to promoters which are improved so as not to undergo methylation in the course of constructing transformants, and utilization thereof.  
     The improved promoters of the present invention are represented by the following (1) and (2):  
     (1) a DNA comprising the nucleotide sequences shown in SEQ ID NOS: 1 to 4; (2) a DNA comprising a nucleotide sequence consisting of the nucleotide sequences shown in SEQ ID NOS 1 to 4, wherein one to several nucleotides are deleted, added or inserted and the deleted, added or inserted sequence is free from any consecutive sequences represented by CG, CAG, CTG, CCG or CGG; the DNA having promoter activity.  
     According to the present invention, the expression efficiency of a structural gene can be enhanced even in a plant, e.g. chrysanthemum, which has weak expression of the structural gene by a cauliflower mosaic virus 35S promoter which has been considered a high expression promoter for plants.

TECHNICAL FILED

[0001] The present invention relates to improved promoters andutilization thereof, in particular to promoters which are improved so asnot to undergo methylation in the course of constructing transformants,and utilization thereof.

DISCLOSURE OF THE INVENTION

[0002] In constructing a desired transformant plant, one of theimportant elements is a high expression promoter. A promoter sequence isa main factor determining transcription level of a gene in plant cells,and in general use of a promoter sequence having a strong transcriptionactivity enables the expression level of target foreign gene to beenhanced. Further, since it becomes remarkably easy to obtain atransformant plant by enhancing expression level of a maker gene, thehigh expression promoter is also important in expressing a drugresistant gene marker for producing a transformant plant.

[0003] Under this circumstance, there have been many reports onobtainment of a high expression promoter in a plant. Typical examplesinclude a cauliflower mosaic virus (CaMV) 35S promoter, and promoters ofan isopentenyl transferase (ipt) gene and a nopaline synthetase (nos)gene of Agrobacterium. Further, some cases have been observed where ahigh expression gene promoter obtained from a genome of a plant which isan object of a transformant host, and is utilized (Genschik et al.,Gene, 148 (1994) 195-202). In recent years, it has been demonstratedthat from among chimeric promoters in which a plurality of thesepromoters are combined, a promoter with remarkably increased promoteractivity can be obtained. By way of example, Min Ni et al. demonstratedthat by combining a promoter of an octopine synthetase (ocs) gene with apromoter of a mannopine (man) synthetase gene derived fromAgrobacterium, a promoter showing high expression in tobacco could beobtained (Plant Journal, 7 (1995) 661-676).

[0004] However, even if these high expression promoters are used, aplant with a required level of expression of a foreign gene cannotnecessarily be obtained in all kinds of plants. One of the reasons forthis may be the existence of specificity based on difference of RNApolymerase existing in each plant cell. Further, as another reason, itis conjectured that a plant has a mechanism to suppress the expressionof the foreign gene. Methylation of cytosine in genome DNA is consideredto be a major factor involved in this gene inactivation or the mechanismfor expression suppression (Meyer and Saedler, Annu. Rev. Plant Physiol.Plant Mol. Biol., 47 (1996) 23-48).

[0005] This methylation of cytosine is known to take place outstandinglyin a double stranded DNA sequence wherein the nucleotide sequences of CGand CNG (N represents any nucleotide) form a palindrome structure(Matzke and Matzke, Plant Physiol. 107 (1995) 679-685). Methylation isalso known to occur even in cytosine of other DNA sequences (Meyer etal., EMBO Journal, 13 (1994) 2084-2088). When cytosine is methylated, itis known that gene expression is suppressed in many organisms (Razin,EMBO Journal, 17 (1998) 4905-4908). In addition, because in a plantgenome, the ratio of methylated cytosine is higher in comparison withother organisms, it has been reported that methylation is closelyrelated to gene inactivation (Meyer and Saedler, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 47 (1996) 23-48). In particular, with respectto a cause for a phenomenon of suppressing the expression of a foreigngene introduced by gene manipulation etc., that is to say “genesilencing”, inactivation by DNA methylation is assumed to be involved.

[0006] However, it has not been reported so far that controllingmethylation of a promoter enhances gene expression. There has been onlyone report on a transient decline of gene expression by forciblymethylating in vitro a gene construct prepared by ligating a CaMV 35Spromoter to a β-glucuronidase (Gus) gene (Hohn et al., Proc. Natl. Acad.Sci. USA, 93 (1996) 8334-8339). However, in the experiment of the abovereport, it was not verified which nucleotide sequence in vivo wassubjected to methylation, and whether the result of that lead to declineof expression. Further, Hohn et al. observed that methylation of notonly a promoter but also cytosine of a structural gene portion causeddecline of expression of the Gus gene, and concluded that it wassignificant for high expression that the structural gene portion shouldnot be subjected to modification with a methyl group. Thus, they seemrather negative toward the idea of high expression by using ademethylation promoter.

[0007] Accordingly, no specific method has been known for enhancing geneexpression level by obviating methylation of a promoter. On top of that,in order to obtain a more highly expressing transformant host byavoiding methylation, it has not been known which CG or CNG sequence ofa promoter portion or a DNA strand including a promoter portion shouldspecifically be modified to what other nucleotides.

DISCLOSURE OF THE INVENTION

[0008] It is an object of the present invention to provide a promoterwhich activates the expression of a structural gene when the promoter isplaced at 5′ side of the structural gene. Further, it is another objectof the present invention to provide a DNA strand comprising thepromoter. Moreover, it is still another object of the present inventionto provide a host which is transformed by the DNA strand and a methodfor high expression of a structural gene by use of the host.

[0009] The inventors, in view of the above-mentioned points, thoughtthat a high expression promoter could be produced by modifying apalindrome DNA sequence consisting of CG and CNG in a double strandedDNA sequence of the promoter to other nucleotides free from CG and CNGsequences without loss of promoter activity and so as to be less likelyto undergo methylation. Based on this idea, as a result of intensivestudies, the inventors found that the expression level of the foreigngene in a chrysanthemum plant could significantly be enhanced byligating a newly designed promoter to a constituent element such as atranslation enhancer, a structural (reporter) gene, a translationtermination codon and a terminator, and by transforming thechrysanthemum plant using the promoter. By this finding, the presentinvention has been accomplished.

[0010] Accordingly, the present invention provides a DNA represented bythe following (a) or (b):

[0011] (a) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:1 from nucleotide No. 7 tonucleotide No. 272; or

[0012] (b) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:1 from nucleotide No. 7 tonucleotide No. 272, wherein one to several nucleotides are deleted,added or inserted at a site other than nucleotide nos. 41 to 42, 59 to60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to 135,145 to 146, 181 to 183, 185 to 186, 197 to 198 and 217 to 218, and thedeleted, added or inserted sequence is free from any consecutivesequences represented by CG, CAG, CTG, CCG or CGG, and the DNA havingpromoter activity.

[0013] Further, the present invention provides a DNA represented by thefollowing (c) or (d):

[0014] (c) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:1; or

[0015] (d) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:1, wherein one to severalnucleotides are deleted, added or inserted at a site other thannucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to110, 119 to 120, 134 to 135, 145 to 146, 181 to 183, 185 to 186, 197 to198 and 217 to 218, and the deleted, added or inserted sequence is freefrom any consecutive sequences represented by CG, CAG, CTG, CCG or CGG,and the DNA having promoter activity.

[0016] Furthermore, the present invention provides a DNA represented bythe following (e) or (f):

[0017] (e) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:2 from nucleotide No. 7 tonucleotide No. 272; or

[0018] (f) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:2 from nucleotide No. 7 tonucleotide No. 272, wherein one to several nucleotides are deleted,added or inserted at a site other than nucleotide nos. 41 to 42, 59 to60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to 135,145 to 146, 181 to 183, 183 to 188, 195 to 200 and 217 to 218, and thedeleted, added or inserted sequence is free from any consecutivesequences represented by CG, CAG, CTG, CCG or CGG (where nucleotide nos.185 to 186 and 197 to 198 are each CG), and the DNA having promoteractivity.

[0019] In addition, the present invention provides a DNA represented bythe following (g) or (h):

[0020] (g) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:2; or

[0021] (h) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:2, wherein one to severalnucleotides are deleted, added and inserted at a site other thannucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to110, 119 to 120, 134 to 135, 145 to 146, 181 to 183, 183 to 188, 195 to200 and 217 to 218, and the deleted, added or inserted sequence is freefrom any consecutive sequences represented by CG, CAG, CTG, CCG or CGG(where nucleotide nos. 185 to 186 and 197 to 198 are each CG), and theDNA having promoter activity.

[0022] Moreover, the present invention provides a DNA represented by thefollowing (i) or (j):

[0023] (i) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:3 from nucleotide No. 7 tonucleotide No. 322; or

[0024] (j) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:3 from nucleotide No. 7 tonucleotide No. 322, wherein one to several nucleotides are deleted,added or inserted at a site other than nucleotide nos. 41 to 42, 59 to60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to 135,145 to 146, 181 to 183, 185 to 186, 197 to 198, 217 to 218, 231 to 233,235 to 236, 247 to 248 and 267 to 268, and the deleted, added orinserted sequence is free from any consecutive sequences represented byCG, CAG, CTG, CCG or CGG, and the DNA having promoter activity.

[0025] Additionally, the present invention provides a DNA represented bythe following (k) or (l):

[0026] (k) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:3; or

[0027] (l) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:3, wherein one to severalnucleotides are deleted, added or inserted at a site other thannucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to110, 119 to 120, 134 to 135, 145 to 146, 181 to 183, 185 to 186, 197 to198, 217 to 218, 231 to 233, 235 to 236, 247 to 248 and 267 to 268, andthe deleted, added or inserted sequence is free from any consecutivesequences represented by CG, CAG, CTG, CCG or CGG, and the DNA havingpromoter activity.

[0028] Further, the present invention provides a DNA represented by thefollowing (m) or (n):

[0029] (m) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:4 from nucleotide No. 7 tonucleotide No. 422; or

[0030] (n) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:4 from nucleotide No. 7 tonucleotide No. 422, wherein one to several nucleotides are deleted,added or inserted at a site other than nucleotide nos. 41 to 42, 59 to60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to 135,145 to 146, 181 to 183, 185 to 186, 197 to 198, 217 to 218, 231 to 233,235 to 236, 247 to 248, 267 to 268, 281 to 283, 285 to 286, 297 to 298,317 to 318, 331 to 333, 335 to 336, 347 to 348 and 367 to 368, and thedeleted, added or inserted sequence is free from any consecutivesequences represented by CG, CAG, CTG, CCG or CGG, and the DNA havingpromoter activity.

[0031] Furthermore, the prevent invention provides a DNA represented bythe following (o) or (p):

[0032] (o) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:4; or

[0033] (p) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:4, wherein one to severalnucleotides are deleted, added and inserted at a site other thannucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to110, 119 to 120, 134 to 135, 145 to 146, 181 to 183, 185 to 186, 197 to198, 217 to 218, 231 to 233, 235 to 236, 247 to 248, 267 to 268, 281 to283, 285 to 286, 297 to 298, 317 to 318, 331 to 333, 335 to 336, 347 to348 and 367 to 368, and the deleted, added or inserted sequence is freefrom any consecutive sequences represented by CG, CAG, CTG, CCG or CGG,and the DNA having promoter activity.

[0034] Moreover, the present invention provides a DNA strand comprisingany of the above DNAs. Such a DNA strand preferably comprises astructural gene DNA and any of the above DNAs which is incorporated at5′ site of the structural gene DNA in a manner such that it isexpressed. These DNA strands may include a constituent element selectedfrom the group consisting of a translation enhancer, a translationtermination codon, a terminator and combinations thereof. Further, thepresent invention provides a host which is transformed by the above DNAstrand. The host is preferably a plant cell.

[0035] Furthermore, the present invention provides a method forexpressing a structural gene in a plant which is characterized in thatthe host transformed by the above DNA strand is cultured or cultivatedso as to enable expression of the structural gene. In this method, thestructural gene may be a foreign gene. In addition, the presentinvention provides a method for producing a protein which is anexpression product of a structural gene whose transcription is activatedor whose expression is promoted by a DNA having promoter activity, usingthe host transformed by the above DNA strand.

[0036] Moreover, the present invention provides a transformant plantwhich is obtained by regeneration from a plant cell transformed by theabove DNA strand.

[0037] Additionally, the present invention provides a DNA strand whichcomprises a selection marker gene DNA and any of the above DNAs whichare incorporated at the 5′ site of the selection marker gene DNA in amanner such that the selection marker gene is expressed. Further, thepresent invention provides a method for selecting a transformant host,which comprises the steps of transforming the host by the DNA strand,and culturing the obtained host under a condition in which the selectionmarker gene can be expressed and it can be identified whether or not thehost expresses the selection marker gene. The host is preferably a plantcell.

[0038] This specification includes part or all of the contents asdisclosed in the specification and/or drawings of Japanese PatentApplication No. 2000-59276, which is a priority document of the presentapplication.

DESCRIPTION OF SEQUENCE LISTS

[0039] SEQ ID NOS: 1 to 4: synthesized DNAs comprising promotersequences

[0040] SEQ ID NOS: 5 to 24: primers

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIGS. 1A and 1B show the comparison between promoters of thepresent invention: a DNA sequence of MF-48 (FIG. 1A) and a DNA sequenceof MF-18 (FIG. 1B) (upper lines respectively), and a DNA sequence of a35S promoter of pBI121 (lower lines).

[0042]FIG. 2 illustrates a portion of structures of plasmids pKT81(A),pKT83(B), pMF-28(C) and pKT11(D).

[0043]FIG. 3 illustrates a plasmid pMF-48-Gus, and its derivativespMF-48-2 and pMF-48-4.

[0044]FIG. 4 shows relative values of averages (among 10 plants or more)of expression level of a Gus gene in chrysanthemum leaves transformed by4 kinds of vectors, based on expression level of the Gus gene inrecombinant tobacco transformed by pBI121 as comparison.

[0045]FIG. 5 shows distribution by plant of relative values (recombinanttobacco transformed by pBI121 was used as comparison) of expressionlevel of the Gus gene in chrysanthemum leaves transformed by 3 kinds ofvectors.

BEST MODE FOR CARRYING OUT THE INVENTION

[0046] (1) Promoter

[0047] A DNA of the present invention is a DNA comprising a nucleotidesequence represented by any of SEQ ID NOS: 1 to 4 or a part thereofhaving promoter activity. These DNAs are prepared by modifying some ofnucleotides in the nucleotide sequences of a CaMV 35S promoter.

[0048] Thirteen CGs or CNGs exist 250 bp upstream from transcriptioninitiation site of the CaMV 35S promoter, and it is estimated that thesenucleotide sequences are methylated by CG methylation enzyme or CNGmethylation enzyme, respectively. Therefore, a DNA in which thesesequences are substituted by sequences which are not subjected tomethylation, is one comprising a nucleotide sequence shown in SEQ IDNO: 1. In SEQ ID NO: 1, the portion having promoter activity is theportion from nucleotide nos. 7 to 272.

[0049] In addition, utilizing methylation-free property of the DNA(promoter) comprising the nucleotide sequence shown in SEQ ID NO: 1, aregion of the promoter, which is called ocs region or as-1 region,comprising about 20 nucleotides (Ellis et al., Plant J., 4 (1993)433-443; Lam et al., Proc. Natl. Acad. Sci. USA, 86 (1989) 7890-7894)can be further modified and this is considered particularly effective.Then, while retaining CG sequences in the ocs region, modification iscarried out to form a palindrome structure of 6 nucleotides (GACGTC),thereby obtaining a DNA comprising a nucleotide sequence shown in SEQ IDNO: 2. The portion of SEQ ID NO: 2 having promoter activity is theportion from nucleotide nos. 7 to 272.

[0050] The comparison between the nucleotide sequences shown in SEQ IDNOS: 1 and 2 (upstream from transcription initiation site), and thesequence of the 35S promoter included in a vector pBI121 (manufacturedby Clontech Company) which is the most commonly used in plant genemanipulation is shown in FIGS. 1A and 1B.

[0051] Moreover, as a modification concerning the ocs region, it isconsidered to conduct synthesis for repeating ocs region in thepromoter, and this is thought to strengthen promoter activity. Thus, ocsregion is repeated such that there are two ocs regions in the SEQ ID NO:1, thereby. obtaining a DNA comprising a nucleotide sequence shown inSEQ ID NO: 3. In SEQ ID NO: 3, a part of nucleotide nos. 7 to 322 haspromoter activity. Further, in SEQ ID NO: 1, ocs regions is repeatedsuch that there are four ocs regions, thereby obtaining a DNA comprisinga nucleotide sequence shown in SEQ ID NO: 4. The portion of SEQ ID NO: 4having promoter activity is the portion from nucleotide nos. 7 to 422.

[0052] The above DNAs of the present invention can be obtained bychemical synthesis in accordance with a method of nucleic acidbiosynthesis.

[0053] Additionally, a DNA of the present invention includes a DNA (avariant) which comprises a nucleotide sequence wherein, relative to anyof the above nucleotide sequences, one to several nucleotides aredeleted, added or inserted, and which has promoter activity. Herein, thenumber of the deleted, added or inserted nucleotides is not particularlylimited, but it is preferably one to several, more preferably one tothree, and most preferably one. Moreover, the DNA of the presentinvention may include a DNA (a variant) which comprises a nucleotidesequence having 80% or more, preferably 90% or more, more preferably 94%or more, and most preferably 99% or more homology with the nucleotidesequence of any of the above DNAs, and which has promoter activity.Herein these homology values are calculated using default parameters(initial settings) by using a nucleotide sequence comparison program:DNASIS-mac v3.7.

[0054] Thus, the variants comprise the nucleotide sequences partiallydifferent from the nucleotide sequences shown in SEQ ID NOS: 1 to 4, butin this case the rearrangement sites from the above CaMV 35S promoterare required to be retained. In other words, in SEQ ID NO: 1, nucleotidenos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119to 120, 134 to 135, 145 to 146, 181 to 183, 185 to 186, 197 to 198, and217 to 218 are not varied. Likewise, in SEQ ID NO: 2, nucleotide nos. 41to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119 to 120,134 to 135, 145 to 146, 181 to 183, 183 to 188, 195 to 200, and 217 to218 are not varied. Further, in SEQ ID NO: 3, nucleotide nos. 41 to 42,59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to135, 145 to 146, 181 to 183, 185 to 186, 197 to 198, 217 to 218, 231 to233, 235 to 236, 247 to 248, and 267 to 268 are not varied. Furthermore,in SEQ ID NO: 4, nucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78,80 to 82, 109 to 110, 119 to 120, 134 to 135, 145 to 146, 181 to 183,185 to 186, 197 to 198, 217 to 218, 231 to 233, 235 to 236, 247 to 248,267 to 268, 281 to 283, 285 to 286, 297 to 298, 317 to 318, 331 to 333,335 to 336, 347 to 348, and 367 to 368 are not varied.

[0055] Moreover, the nucleotide sequences of the above variants arerequired to be free from any consecutive sequences represented by CG,CAG, CTG, CCG or CGG, but the nucleotide nos. 185 to 186 and 197 to 198in SEQ ID NO: 2 are each exceptionally CG.

[0056] As long as the DNAs comprising the nucleotide sequences shown inthese SEQ ID NOS: 1 to 4 or the variants thereof have promoter activity,the activity level thereof is not particularly limited, but it ispreferable to substantially retain promoter activity of the DNAscomprising the nucleotide sequences shown in SEQ ID NO: 1 to 4 orpromoter activity of the parts thereof. Here “substantially retainingpromoter activity” of these DNAs or the parts thereof means, in apractical example using promoter activity, to retain almost the sameusable level of activity under the same condition as these DNAs and theparts thereof. Further, the promoter activity described herein isdefined as an activity preferably in plant cells, more preferably inchrysanthemum plants, and most preferably in a chrysanthemum cultivar,Reagan (Chrysanthemum morifolium cv. Reagan or Dendranthema grandiflorumcv. Reagan).

[0057] It is obvious that these variants can be selected and preparedwithout any special difficulty by a person, as long as the person isskilled in the art, with reference to the nucleotide sequences shown inSEQ ID NOS: 1 to 4 in accordance with the descriptions in literaturesuch as Molecular Cloning (edited by Sambrook et al. (1989) Cold SpringHarbor Lab. Press, New York). Further, a person skilled in the art canobtain and utilize the variants with respect to a technique forartificially replacing, deleting, inserting or adding one or morenucleotides from the nucleotide sequences relative to the nucleotidesequences (site-specific derivation of mutation) shown in theabove-mentioned SEQ ID NOS: 1 to 4, in accordance with techniquesdescribed in Proc. Natl. Acad. Sci. USA 81(1984) 5662-5666, WO85/00817,Nature 316(1985) 601-605, Gene 34(1985) 315-323, Nucleic Acids Res.13(1985) 4431-4442, Proc. Natl. Acad. Sci. USA 79(1982) 6409-6413,Science 224(1984) 1431-1433, etc.

[0058] It can be verified according to a method for promoter activitydetermination as described below, whether or not the above obtainedvariants have promoter activity, and further whether or not theysubstantially have promoter activity of any of the DNAs comprising thenucleotide sequences shown in SEQ ID NO: 1 to 4 or the parts thereof.

[0059] The promoter activity of the above variants can be calculatedpreferably by preparing a vector having various reporter gene, such asgenes of β-glucuronidase (Gus), luciferase (Luc), chloramphenicolacetyltransferase (Cat), β-galactosidase (Gal), nopalin synthetase(nos), octopine synthetase (ocs) etc. (“Plant genetic transformation andgene expression; a laboratory manual”, edited by Draper, J. et al.,Blackwell Scientific Publication, 1988) ligated to the downstream regionof the novel promoter; inserting the vector into plant cell genome withvarious transforming methods (described later) which are conventionallywell known and commonly used; and measuring expression level of thereporter gene, but it is not limited to this method. As one examplethereof, in the case where a reporter gene is Gus, promoter activity inhost cells is determined in accordance with (i) a histochemical Gusstain method and/or (ii) a method using fluorescent substrate (bothmethods are in Plant Molecular Biology Manual, C2 (1994) 1-32 (Ed.)Gelvin and Schilperoort, Kluwer Academic Publishers). Moreover, amountsof protein were measured in accordance with, for example, the Bradfordmethod (Anal. Biochem. 72 (1976) 248-254), and Gus activity wasconverted to a value per amounts of protein (for example, calculated aspmoleMU/min/mg protein) for determination of promoter activity.

[0060] Exemplary host cells to be preferably used for the DNA of thepresent invention are cells of various plants such as monocotyledonsincluding rice, MUGI (general name of wheat, barley, rye and oat), corn,onion, lily, orchid, etc. and dicotyledons including soy bean, rapeseed,tomato, potato, chrysanthemum, rose, carnation, petunia, gypsophila,cyclamen, etc. Particularly preferable examples include cells of a plantsuch as chrysanthemum which has high chromosomal polyploidy. The reasonfor that is that a plant is considered to methylate genes forinactivating homologous genes, and since plants with high polyploidyhave many homologous genes it is expected that these genes will beinactivated by a strong methylation mechanism. (Leitch and Bennett,Trends in Plant Sci., 2 (1997) 470-476) In addition, because of a highCG content of the genome (Thomas and Sherratt, Biochem. J., 62 (1956)1-4), cells of plants having target sequences of methylation reaction inabundance are also preferable candidates.

[0061] (2) DNA Strand

[0062] According to the present invention, there is provided a DNAstrand comprising the DNA of the present invention. Such a DNA strandcan be used for transcribing any gene, and for use thereof a desiredgene is incorporated into the DNA strand in an expressible form. Such agene is typically a structural gene. Accordingly, the present inventionfurther provides a structural gene DNA and a DNA strand comprising theDNA of the present invention which is incorporated at the 5′ site of thestructural gene DNA in a manner so as to express the structural gene.

[0063] A specific example of the DNA strand according to the presentinvention may be, for example, where the DNA of the present invention isinserted as a part of constituent element in a plasmid or a phage DNA.

[0064] When incorporating the structural DNA gene into such a DNAstrand, the DNA or the structural DNA of the present invention can bearranged to enable expression of the structural gene. Examples of thestructural gene DNA include β-glucan elicitor receptor (Umemoto et al.,Proc. Natl. Acad. Sci. USA 94 (1997) 1029-1034), pac1 (Sano et al.,Biotechnol. 15 (1997) 1290-1294) and a DNA encoding 2-5Aase or RNaseL(Ogawa et al., Natl. Biotechnol. 14 (1996) 1566-1569), but it is notlimited to these.

[0065] The DNA strand of the present invention may further include aconstituent element such as a translation enhancer, a translationtermination codon, a terminator, etc. As a translation enhancer,translation termination codon, and terminator, known ones can be used ina suitable combination. Examples of a virus-originated translationenhancer include sequences of tobacco mosaic virus, alfalfa mosaic virusRNA4, bromomosaic virus RNA3, potato virus X, tobacco etch virus, etc.(Gallie et al., Nuc. Acids Res., 15 (1987) 8693-8711) Further, examplesof a plant-originated translation enhancer include sequences derivedfrom soy bean β-1,3-glucanase (Glu) (Isao ISHIDA, Norihiko MISAWA,edited by Kodansha Scientific, “Saibo-kogaku-jikkenn-sousa-nyumon”(Introductory for operation in cell engineering experiments), KodanshaLtd., p.119, 1992), and sequences derived from ferredoxin affinitysubunit (PsaDb) of tobacco (Yamamoto et al., J. Biol. Chem., 270 (1995)12466-12470). Examples of the terminator include terminators of nosgene, ocs gene, etc. (Annu. Rev. Plant Physiol. Plant Mol. Biol., 44(1993) 985-994, “Plant genetic transformation and gene expression; alaboratory manual” described before). Moreover, it has been reportedthat activity can be enhanced by identifying the 35S enhancer part as atranscription enhancer in a promoter and ligating a plurality of them toeach other in series (Plant Cell, 1 (1989) 141-150). This part can beused as a part of the DNA strand. These various constituent elements arepreferably incorporated into the DNA strand in a form so as to functionin accordance with their characters. A person skilled in the art canappropriately conduct such a manipulation.

[0066] The DNA (promoter) of the present invention includes atranslation enhancer derived from Glu gene of soy bean aftertranscription initiation site (described as after nucleotide nos. 279 inSEQ ID NOS: 1 and 2, after nos. 329 in SEQ ID NO: 3, or after nos. 429in SEQ ID NO: 4). Although this sequence is not directly related intranscriptional promotion due to its being methylation-free, it canfurther promote expression of a target gene. The translation enhancer isnot limited to the one used in the present invention which is derivedfrom Glu gene of soy bean, but the same effect can be expected evenwhere it is replaced with an other translation enhancer such as theabove which have been so far reported. Moreover, as with the presentinvention, the CG sequence of this translation enhancer can be replacedwith other nucleotides. In addition, when the other translation enhancerhas a CNG sequence other than a CG sequence, the CNG sequence can bereplaced with other nucleotides in the same manner as with the CGsequence. A person skilled in the art can appropriately conduct such amodification.

[0067] The above DNA strand can easily be prepared by a person skilledin the art using a method which is commonly used in the field of geneengineering. Further, the DNA strand of the present invention is notlimited to an artificial construct, and as long as it has a structuresuch as the above, it may be isolated from a natural source. The DNAstrand can be obtained by synthesis according to a well-known andcommonly used method of nucleic acid biosynthesis.

[0068] (3) Transformation

[0069] The DNA strand comprising the DNA having promoter activity of thepresent invention enables a host to be transformed, and the obtainedtransformant is cultured or cultivated, thereby allowing induction ofexpression of the structural gene, or expression of the structural genewith high efficiency. The structural gene may be a foreign gene.

[0070] The chain of the present invention after the transformation canbe present in microorganisms (especially bacteria), phage particles orplants in a form of incorporation into plasmids, phages or genome DNA.Here typical examples of the bacteria include, but are not limited to,Escherichia coli, and Agrobacterium, etc.

[0071] In a preferable example of the present invention, the DNA strandof the present invention exists in plants in a form such that the DNA(promoter) of the present invention, the translation enhancer, thestructural gene DNA, the translation termination codon, the terminator,etc. are integrally ligated and incorporated into a genome so that thestructural gene which attempts to express a protein can be stablyexpressed in plants.

[0072] Preferable examples of the host include cells of monocotyledonssuch as rice, MUGI(general name of wheat, barley, rye and oat), corn,onion, lily, orchid, etc. and dicotyledons such as soy bean, rapeseed,tomato, potato, chrysanthemum, rose, carnation, petunia, gypsophila,cyclamen, etc., and in particular preferable examples are cells ofplants such as chrysanthemum, etc. having a high chromosomal polyploidy.In addition, exemplary plant materials include growing points, shootprimordial meristems, leaf pieces, stem pieces, root pieces, tuberpieces, petiole pieces, protoplast, calli, anthers, pollen, pollentubes, peduncle pieces, scape pieces, petals, sepal pieces, etc.

[0073] As a biological method for introducing a foreign gene into thehost, various methods which have already been reported and establishedcan be used as appropriate. Preferable examples thereof include a methodwherein Ti plasmid, Ri plasmid, etc. of virus or Agrobacterium are usedas a vector, physical methods for introducing a gene withelectroporation, polyethylene glycol, particle gun, micro-injection(“Plant genetic transformation and gene expression; a laboratory manual”described before), silicon nitride whisker, silicon carbide whisker(Euphytica 85 (1995) 75-80, In Vitro Cell. Dev. Biol. 31 (1995) 101-104,Plant Science 132 (1998) 31-43), and the like. A person skilled in theart can appropriately select and utilize a method of introduction.

[0074] Furthermore, a transformant plant which expresses the introducedgene in its cells can be prepared by regenerating the plant cells whichhave been transformed with the DNA strand of the present invention. Thismanipulation can easily be carried out by a person skilled in the artwith a generally known method of regenerating plant cells to plants.Regarding regeneration of plant cells to plants, see literature, forexample “SHOKUBUTU-SAIBOU BAIYOU MANUAL (Manual for Plant CellCultivation) (edited by Yasuyuki Yamada, Kodansha Scientific, 1984).

[0075] When an expression product of a gene which has beenexpression-induced or highly expressed, is desired for use as anisolated product, it can be isolated and purified from the cultureaccording to an appropriate method depending on the expression product.The expression product of the target structural gene can be highlyexpressed by culturing such a host when the growth of a host cell andfurther properties of the cell are altered with the existence of theexpression product, or by cultivating such a plant when the host is adedifferentiated plant.

[0076] Moreover, the high expression promoter disclosed in the presentinvention permits remarkable enhancement of the efficiency of planttransformation by using a DNA strand expressing a selection marker ofe.g. kanamycin resistant (e.g. NPTII) gene. This can be accomplished byusing a selection marker gene DNA and a DNA strand comprising any of theabove DNAs which is incorporated at the 5′ site of the selection markergene DNA in a form so as to express the selection marker gene. Aspecific procedure therefor is not particularly limited, but theprocedure can be accomplished by e.g. transforming a host with the DNAstrand, and culturing the obtained host under the conditions where theselection marker gene can be expressed, and whether or not the hostexpresses the selection marker gene can be determined. The host may beother than plant cells, so it is not particularly limited, but it ispreferably a plant cell. Herein any selection marker gene can be used,so it is not particularly limited, but it is preferably a drug-resistantgene, e.g. NPTII gene (kanamycin resistant gene), Hyg^(r) gene(hygromycin resistant gene), is usable. In this case, the condition fordetermining whether or not the host can express the selection markergene, is attained by culturing it with a medium containing a drug towhich the gene is resistant. A drug to be used may be kanamycin (Km)when NPTII gene is used as a selection marker gene, but it is notlimited thereto, and G418 or paromomycin may be optionally selected foruse. Further, the same effect can be expected when a DNA strandexpressing e.g. a selection marker of hygromycin resistant (e.g.Hyg^(r)) gene is used. A drug used in this case may be hygromycin.

[0077] In this case also, as a method for obtaining a transformed plantby introducing the DNA strand into the host cells, any of the methodsmentioned above can be used. Confirmation of the expression of theplant, when e.g. a reporter gene is Gus, can be carried out by the above(i) histochemical Gus stain method and/or (ii) a method usingfluorescent substrates (Plant Molecular biology Manual, C2 (1994) 1-32,described before) etc.

[0078] In general, during cell differentiation, it is known that geneinactivation occurs by methylation. The promoter disclosed in thepresent invention is expected to cause high expression of a target genenot only in undifferentiated plant cells such as callus attransformation, but also in a plant.

[0079] Industrial Applicability

[0080] According to the present invention, expression efficiency can beenhanced in a plant which shows weak expression of a structural gene bycauliflower mosaic virus 35S promoter conventionally regarded as a highexpression promoter for plants, e.g. chrysanthemum.

EXAMPLES

[0081] The present invention will hereinafter be described withreference to the following Examples, but it is not limited thereto.

Example 1

[0082] Preparation of a Methylation Free Promoter

[0083] Thirteen CGs or CNGs exist 250 bp upstream from the mRNA(transcription) initiation site of a CaMV 35S promoter, and it isconjectured that these nucleotide sequences are subjected to methylationin plants by CG methylase and CNG methylase respectively. Accordingly, aDNA was synthesized in its entirety, by substituting these sequences bysequences which are not subjected to methylation. (MF-48: SEQ ID NO:1)Further, with respect to ocs region which is considered to have largeinfluence especially on transcriptional activity, while CG sequencestherein were retained, a DNA was prepared by modification so as to havea palindrome structure of 6 nucleotides (GACGTC). (MF-18: SEQ ID NO:2) Acomparison between the sequences (upstream from the transcriptioninitiation site) of MF-48 and MF-18 and the sequence of a 35S promoterincluded in a vector pBI121 which is usually most often used for plantgene manipulation, is shown in FIGS. 1A and 1B. In FIGS. 1A and 1B, theunderlined parts indicate CG and CNG sequences which are said to betargets for methylation.

[0084] Besides the above, a gene (plasmid name is pSan9: Proc. Natl.Acad. Sci. USA, 93 (1996) 8334-8339, described before) havingmethylation target sites of 35S promoter which has been modified wastransferred from Dr. Hohn of Friedrich Miescher-Institut (Switzerland).This gene was composed of a promoter portion of about 250 nucleotidepairs upstream of mRNA transcription initiation site, a 5′non-translation sequence of about 50 nucleotide pairs of a CaMV 35S genedownstream thereof (hereinafter the combination of the promoter portionand the 5′ non-translation sequence is referred to as “MF-28”), and aGus gene expression cassette further downstream thereof comprising a Gusstructural (reporter) gene and a 35S terminator.

Example 2

[0085] Preparation of a Vector Having a Methylation Free Promoter

[0086] In order to confirm the effect of the thus prepared MF-48, MF-18and MF-28 in a transformed plant, expression vectors were constructed.The following expression vectors were all composed of expression unitsof a Gus gene and an NPTII gene within a region flanked by bordersequences so that the transcription directions thereof were inopposition. For these expression cassettes, a binary type vector, pKT11which was amplifiable with Agrobacterium and Escherichia coli, was usedas a basic vector, and the promoters of the Gus gene and NPTII gene wasreplaced, thereby constructing pKT81, pKT83 and pMF-28.

[0087] The binary type vector pKT11 was a vector composed of aXhoI-EcoRI portion about 250 bp which was an RB region of AgrobacteriumA281, an HindIII-EcoRI portion about 3.5 kbp (ligating from 5′ side inorder of a CaMV 35S promoter, a translation enhancer of tobacco PsaDb,the Gus gene comprising an intron of a ricinus catalase gene and a nosgene terminator) which was an expression unit portion of the Gus gene, aHindIII-KpnI portion about 1.7 kbp (terminators of NPTII gene and nosgene which function in a plant driven by a nos gene promoter) which wasan expression unit portion of the NPTII gene, and a KpnI-XhoI portionabout 5.5 kbp which was a portion having a left border region derivedfrom pBI121 and a replication origin amplifiable with Agrobacterium andE. coli. Further, it is a vector in which Gus gene is expressed by apromoter to which a translation enhancer of a soybean derived Glu geneis ligated (FIG. 2D).

[0088] The DNA fragments (promoter) prepared in Example 1 were purifiedby agarose gel, and the 35S promoter region (HindIII-XbaI) of theplasmid pKT11 was substituted, thereby preparing plasmids pKT81 andpKT83 (FIG. 2).

[0089] Each of pKT81 and pKT83 has a translation enhancer of soybeanderived Glu gene derived from soybean, which is ligated downstream ofMF-48 and MF-18 respectively, for the purpose of enhancing translationefficiency, and further downstream has an expression unit in which theGus gene which was a reporter gene and the nos gene terminator areligated. Simultaneously, each of them includes a expression unit inwhich an ipt gene promoter, a translation enhancer of Glu gene derivedfrom soybean, an NPTII gene and an nos gene terminator are ligated.

[0090] In pMF-28, the entire Gus gene expression cassette of pSan9 wasexchanged with Gus expression cassette of pKT11.

[0091] Restriction enzyme maps of vectors pKT81, pKT83 and pMF-28 whichexpress Gus gene by promoters of MF-48 and MF-18, and MF-28 are shown inFIGS. 2A, B and C, and pKT11 to be used as a basal vector is shown inFIG. 2D.

[0092] In addition, in order to prepare a promoter having increased ocsregions, the sequence between excision sites of each restriction enzymeBamHI and BglII, which is a region including ocs element of MF-48promoter, was inserted into the fragment prepared by excising thepromoter with BamHI, and there were prepared a promoter (MF-48-2: SEQ IDNO:3) having two copies of this sequence aligned in series in the samedirection and a promoter (MF-48-4: SEQ ID NO:4) having four copies ofthis sequence aligned in series in the same direction. (FIG. 3)

Example 3

[0093] Preparation of Chrysanthemum Callus which Expresses Gus Gene

[0094] Vectors pKT81 and pKT83 which express Gus gene by promoters ofMF-48 and MF-18, and a vector pKT11 as a control were each introduced byelectroporation into Agrobacterium tumefaciens LBA 4404 strain, theobtained strain was inoculated in 3 ml of YEB-Km medium, and it wascultured for 16 hours at 28° C. in the dark. Thereafter, strain cellswere collected by centrifugation, and suspended in 10 ml of thefollowing infected medium, preparing the infected solution. Thecompositions of YEB-KM medium and the infected medium are as follows.

[0095] YEB-Km medium; 5 g/l beef extract, 1 g/l yeast extract, 5 g/lpeptone, 5 g/l sucrose, 2 mM magnesium sulfate (pH 7.2), and 50 mg/lkanamycin

[0096] Infected medium; inorganic salts and vitamins of ½ concentrationof MS (Murashige & Skoog, Physiol. Plant., 15 (1962) 473-497) medium, 15g/l sucrose, 10 g/l glucose, and 10 mM MES (pH 5.4)

[0097] Leaves from a sterile plant of Reagan (Chrysanthemumn morifoliumcv. Reagan or Dendranthema grandiflorum cv. Reagan), a cultivar ofchrysanthemum, were cut into leaf pieces of 5 to 7 mm square, and thepieces were each dipped for 10 minutes into Agrobacterium infectedsolutions into which each of vectors pKT11, pKT81 and pKT83 wasintroduced. After wiping off any excess of the infected solution with apaper filter, the leaf pieces were transferred to the followingco-cultivation medium and cultivated at 25° C. in the dark. Afterthree-day cultivation, the leaf pieces were transferred to the followingselection medium for three-week cultivation, thereby obtainingKm-resistant calli. Cultivation in the selection medium was conductedwith conditions of 16-hour lighting (light density of 32 μE/m²s) /8-hour non-lighting at 25° C. Four leaf pieces containing the obtainedKm resistant callus for each vector, in total 12 leaf pieces, were usedfor Gus activity determination in order to confirm Gus gene expression.

[0098] Co-cultivation medium; inorganic salts and vitamins of MS medium,30 g/l sucrose, 1 mg/l naphthalenacetic acid, 2 mg/l benzyladenine, 8g/l agar, 5 mM MES (pH 5.8), and 200 μM acetosyringone

[0099] Selection medium; inorganic salts and vitamins of MS medium, 30g/l sucrose, 1 mg/l naphthalenacetic acid, 2 mg/l benzyladenine, 8 g/lagar, 5 mM MES (pH 5.8), 25 mg/l kanamycin, and 300 mg/l cefotaxime

[0100] The leaf piece was transferred to 200 μl of a reaction solution(100 mM sodium phosphate buffer solution (pH 7.0), 1 mM EDTA, 0.1%Triton X-100, 1 mM dithiothreitol (DTT)) for determination of enzymeactivity, and fully crushed while ice-cooling. The obtained suspensionwas centrifuged to collect a supernatant, and the supernatant was usedas crude enzyme solution. The Gus activity determination was conductedin accordance with the published report (Plant Molecular Biology Manual,C2 (1994) 1-32, described before). Namely, 5 μl of the crude enzymesolution and 50 μl of 2.8 mg/ml 4-methylumbelliferyl-β-D-glucuronide asa substrate were added to 145 μl of the reaction solution, and thegenerated fluorescence was measured. The measurement of amounts ofprotein was conducted using Protein Assay Kit II of Bio-RadLaboratories, and Gus activity per amounts of protein were determined.The following Table 1 shows Gus activity of calli which were transformedby pKT11, pKT81 and pKT83 respectively. Since the calli transformed bypKT81 and pKT83 exhibited about 4 to 5 times higher Gus activity thanthe callus transformed by pKT11, it was confirmed that methylation-freepromoters: MF-48 and MF-18 have high expression ability. TABLE 1Expression of β-glucuronidase gene in chrysanthemum callus by variouspromoters Vector β-glucuronidase (Gus) activity (Promoter)(pmole/min./mg protein) pKT11(35S) 4.43 pKT81(MF-48) 23.69 pKT83(MF-18)15.71

[0101] With respect to MF-48-2 and MF-48-4, vectors were prepared byreplacing 35S promoters of pBI121, and in the same manner as above,using chrysanthemum leaves as material, transformation was conducted,thereby obtaining Km resistant calli. Using these calli, in accordancewith the method described in Plant Molecular Biology Manual, C2 (1994)1-32 (described before), a tissue stain process was conducted with Gusactivity. Color development was not observed with pBI121, but incontrast remarkably strong blue color development was observed withMF-48-2 and MF-48-4. In other words, with regard to qualitativeexpression strength, pBI121 was negative (−), but in contrast MF-48-2and MF-48-4 were positive (+) to (++).

Example 4

[0102] Preparation of a Chrysanthemum Plant which Expresses Gus Gene

[0103] In plant transformation, it is often observed that even though apromoter can be highly expressed in undifferentiated cells like callus,that promoter can be highly expressed in only a few cells of grownplants. As one of the reasons for this phenomenon, it is thought thatgene methylation is stimulated to suppress unnecessary gene expressionwhen the plant cells differentiate. In view of the foregoing, we thoughtthat a methylation-free promoter disclosed in the present invention canbe highly expressed in grown plants, too, and thus we examined this.

[0104]Agrobacterium tumefaciens LBA 4404 strains each containing pKT81,pKT83, and pMF-28 were transformed in accordance with the method ofExample 3, and Km resistant calli were obtained. From the obtainedcalli, plants were regenerated on MS medium containing Km (regenerationmedium; the composition was the same as the selection medium of Example3). Further, for facilitating rooting, the regenerated plants were grownon a rooting-facilitating medium wherein plant growth regulatingsubstances (naphthalenacetic acid, benzyladenine) were removed from theregeneration medium.

[0105] From the grown plants, plants containing an NPTII gene as aforeign gene were detected by conducting PCR, and it was confirmed thatthe thus-obtained redifferentiated plants were transformants. Here as aprimer for specific amplification of an NPTII gene-specific sequence,the sequences of TAAAGCACGAGGAAGCGGT (SEQ ID NO:5) andGCACAACAGACAATCGGCT (SEQ ID NO:6) were used. The reaction conditions forPCR were heating at 94° C. for 5 minutes; 30 cycles of 30 seconds at 94°C., 1 minute at 55° C., and 1 minute at 72° C.; and thereafter areaction at 72° C. for 10 minutes. In this reaction, ExTaq polymelase(manufactured by Takara Shuzo Co., Ltd.) was used as an enzyme.

[0106] With respect to 3 leaves of each of these plants and 10 plantsper each gene, the activity was determined in the same manner as Kmresistant callus, and mean values of the determined activity are shownin FIG. 4. As a comparison, in accordance with the transformation methodof Example 3, the experiments for introducing pBI121 into chrysanthemumand tobacco (variety: Xanthi) were conducted. Further, leaves ofchrysanthemum transformants each having pKT81(MF-48), pKT83(MF-18), andpMF-28(MF-28) introduced thereinto, were examined for measuringexpression level of the Gus gene by a plant, and the results thereof areshown by a histogram in FIG. 5.

[0107] According to the results, it was observed that chrysanthemumshaving pKT81(MF-48), pKT83(MF-18), or pMF-28(MF-28) introduced thereintoeach exhibited higher expression of the Gus gene than one having pBI121.However, the expression level of pMF-28 did not reach the expressionlevel in tobacco which is well known as example of a high expressionpromoter. Nevertheless, surprisingly, pKT81 and pKT83 exhibited muchhigher expression level than pBI121(35S) in tobacco. Then, whenpKT81(MF-48) and pKT83(MF-18) were compared with each other,pKT83(MF-18) plants tended to exhibit higher expression of the Gus gene.(FIG. 4) In addition, the expression level in chrysanthemum by a plantwas studied. For example, plants which exhibited more than three timesthe expression level of the Gus gene were limited to MF-48 and MF-18. Asa whole, the number of plants exhibiting high expression was likely tobe larger in the case of MF-18, compared with MF-48. (FIG. 5)

Example 5

[0108] Analysis of Foreign Gene Methylation in Transformed Chrysanthemum

[0109] In order to understand how the introduced gene is methylated invivo, the location of methylated cytosine in the introduced genesequence was determined. The analytical method to be used was cytosinedeamination PCR method of Meyer et al. (EMBO Journal, 13 (1994)2084-2088). An outline of the method is that firstly a DNA was extractedby CTAB from transformed chrysanthemum obtained in Example 4 and excisedby a restriction enzyme EcoRI, suspended in a conversion buffer (3MNa-bissulfate, 0.5 mM hydroquinone, pH 5.3), and reacted at 50° C. for20 hours in a nitrogen gas phase. After desalting by dialysis, DNA wasalkali-denatured with 0.3N NaOH, and then precipitated by ethanol forcollection. Next, using DNA primers designed to flank nucleotidesequence-determining site, PCR reaction was conducted. The PCR primersused herein are shown as follows.

[0110] <Primer for Analysis of 35S Region Methylation>

[0111] First PCR (SEQ ID NO:7) 35S-8:GAATGTTAATTTATAGATGGTTAGAGAGGTTTATGTAGTAGG (SEQ ID NO:8) 35S-8:CCATATTCTCTCCAAATAAAATAAAC

[0112] Second PCR (SEQ ID NO:9) 35S-9:AGTAATAATTTTAGGAAATTAAATATTTTTTTAAGAAGG (SEQ ID NO:10) 35S-14:TATTCTCTCCAAATAAAATAAACTTC

[0113] <Primer for Analysis of 35S Complementary Chain Methylation>

[0114] First PCR (SEQ ID NO:11) 35S-C-1: CTATTCCAATATAAACAATTCAAAACTTAC(SEQ ID NO:12) 35S-C-4: TGAAATGAATTTTTTTATATAGAGGAAGGGTTTTGTG

[0115] Second PCR (SEQ ID NO:13) 35S-C-2:CAACATAATAAAACACAACACACTTATCTAC (SEQ ID NO:14) 35S-C-3:ATGAATTTTTTTATATAGAGGAAGGGTTTTGTGAAG

[0116] <Primer for Analysis of GUS Gene Methylation>

[0117] First PCR 35S-16: GAAGAAATTTTTGTTAATATGGTGGAGTATGATATG (SEQ IDNO:15) TO-100: CCAATCAACAAACACATAATTACAATCTTACACAACATACATC (SEQ IDNO:16)

[0118] Second PCR (SEQ ID NO:17) 35S-17:GGGATGATGTATAATTTTATTATTTTTTGTAAGA (SEQ ID NO:18) TO-101:CATAACATCAACTTCAAATAACATATAACCACCCTAATAC

[0119] <Primer for Analysis of Pac1 Gene Methylation>

[0120] First PCR (SEQ ID NO:15) 35S-16:GAAGAAATTTTTGTTAATATGGTGGAGTATGATATG (SEQ ID NO:19) pac1-7:CTTCAATAACAAATTCATTTTAACAATCATACC

[0121] Second PCR (SEQ ID NO:17) 35S-17:GGGATGATGTATAATTTTATTATTTTTTGTAAGA (SEQ ID NO:20) pac1-8:ACAAATTCATTTTAACAATCATACCTTAACT

[0122] <Primer for Analysis of MF Methylation>

[0123] First PCR TO-103: GAGGATTTAAAAGGAAGGTGGTTTTTATAAATGTTATTATTG (SEQID NO:21) TO-105: CCACAATTTTCACAATCCAAACTAAATACCCACAAACC: GUS gene5′ region (SEQ ID NO:22)

[0124] Second PCR TO-104: GGATTTAAAAGGAAGGTGGTTTTTATAAATGTTATTATTGTG(SEQ ID NO:23) TO-106: CAATTTTCACAATCCAAACTAAATACCCACAAACCATC: GUS gene5′ region (SEQ ID NO:24)

[0125] The reaction conditions for all the above PCR were initialheating at 94° C. for 5 minutes; 30 cycles of 30 seconds at 94° C., 1minute at 65° C., and 1 minute at 72° C., followed by a reaction at 72°C. for 10 minutes. Secondly, using 1 μl of the firstly obtained PCRproduct, after carrying out heating at 94° C. for 5 minutes, a cycle of30 seconds at 94° C., 1 minute at 60° C., and 1 minute at 72° C. wasrepeated 30 times. Finally a reaction at 72° C. for 10 minutes wascarried out.

[0126] In this reaction, ExTaq polymelase (manufactured by Takara ShuzoCo., Ltd.) was used as an enzyme, and the PCR synthesis product wascloned into pT7blue. With regard to the original DNA, each DNA sequenceon about 5 clones was determined. According to a series of theseconversion reactions, cytosine was converted to uridine, and methylatedcytosine was read as cytosine per se, thereby identifying the existenceof a methyl group. The difference in nucleotide sequences was analyzedby DNASIS-Mac v3.7.

[0127] About 10 plants of chrysanthemum transformants obtained inExample 4 were analyzed. As a result, in a chrysanthemum transformantexhibiting high expression of the Gus gene, there were relatively fewmethylated cytosines in the 35S promoter, and methylation ratio in about5 clones was also low. On the other hand, in the plants from which noexpression of the Gus gene was detected, almost all the cytosinesincluding CG and CNG in the 35S promoter were methylated at a highratio. Here, as comparative experiment, with regard to tobacco (variety:Xanthi) having the same gene introduced thereinto, plants thereofexhibiting high expression of the Gus gene were investigated, and nomethylated cytosine was detected. Incidentally, methylation degree ofcytosine was investigated regarding a complementary DNA strand to the35S promoter in chrysanthemum. Although there were scattered methylatedcytosines, specific cytosines were not modified and there was nocorrelation with Gus gene expression. On the other hand, methylation ofa structural gene was also investigated and cytosine modification washardly observed at least from the translation initiation site (ATG) tonucleotide no. about 600, regardless of expression strength. The aboverelationship between cytosine methylation and expression level wasobserved not only in Gus gene but also in chrysanthemum havingintroduced thereinto a structure wherein a double-stranded RNA specificRNase gene (Nature Biotechnoloty, 15, 1290-1297 described before) wasligated downstream of the 35S promoter.

[0128] Next, methylation was investigated in the same manner concerningchrysanthemum having pKT81 introduced thereinto and exhibiting highexpression of the Gus gene. As a result, by transformation, some plantswere not subjected to methylation at all, and in the other plantscytosines at a site other than palindrome structure were stronglymethylated from the promoter to the Gus gene. However, all groups ofplants strongly expressed the Gus gene, and because of this, methylationof cytosine residues other than the palindrome structures (CG and CNG)was considered to exert a little influence on gene expression.

[0129] Accordingly, as one method for enhancing structural geneexpression, it was considered important to convert nucleotide sequencehaving a palindrome structure of CG and CNG at the site of the promoterof the introduced gene. In passing, among palindrome structures, in thecase of pKT83, though CG sequence (nos. 185 and 197 cytosine nucleotidein SEQ ID NO:2) in a short sequence, so-called ocs sequence has asequence not to be converted, the effect of this site had a tendency toshow higher activity than pKT81 according to the results of Example 3(FIGS. 4 and 5). Thus, to prepare a high expression promoter, withoutconversion of the palindrome structure of this ocs sequence, it wasindicated that much higher expression in vivo can be accomplished.

Example 6

[0130] Transformation of Carnation

[0131] For the purpose of increasing the expression level of an NPTIIgene by a methylation-free promoter and enhancing selection efficiencywith a marker, a Nos promoter of NPTII expression cassette was changedto the methylation-free promoter. To put it simply, the Nos promoter andthe MF-48 promoter in the cassette were replaced with a DNA fragmentbetween HindlIl and XbaI, thereby preparing a cassette of MF-48promoter, NPTII and Nos terminator. By replacing the NPTII expressioncassette (HindIII to KpnI) in pKT-11 with the obtained cassette, pKT74was prepared as a binary vector ligated to Gus expression cassette.

[0132] This pKT74 and, as a control, pKT11, were introduced intoAgrobacterium tumefaciens LBA 4404 strains by electroporation, and thesewere inoculated on 3 ml of YEB-Km medium. After cultivation at 28° C.for 16 hours, strain cells were collected by centrifugation andsuspended in 10 ml of the following infected medium, thereby preparingan infected solution. The compositions of the YEB-Km medium and theinfected medium were as follows.

[0133] YEB-Km medium; 5 g/l beef extract, 1 g/l yeast extract, 5 g/lpeptone, 5 g/l sucrose, 2 mM magnesium sulfate (pH 7.2), and 50 mg/lkanamycin

[0134] Infected medium; inorganic salts and vitamins of ½ concentrationof MS (Murashige & Skoog, Physiol. Plant., 15 (1962) 473-497), 15 g/lsucrose, 10 g/l glucose, and 10 mM MES (pH 5.4)

[0135] Leaf stems were cut off from sterile plants of carnationcultivar, Scania (Dianthus caryophyllus L), and they were dipped for 10minutes into Agrobacterium infected solutions each having introducedthereinto pKT11 and pKT74. After wiping off any excess of the infectedsolution with a paper filter, they were transplanted in the followingco-cultivation medium and cultivated at 25° C. in the dark. Afterthree-day cultivation, they were transplanted in the following selectionmedium and cultivated for 3 weeks, thereby obtaining G418 resistantcalli. The cultivation in the selection medium was conducted with theconditions of 16-hour lighting (light density 32 μE/m²s)/8-hournon-lighting at 25° C.

[0136] Co-cultivation medium; inorganic salts and vitamins of MS medium,30 g/l sucrose, 0.5 mg/l indolebutyric acid, 0.22 mg/l thidiazuron, 8g/l agar, 5 mM MES (pH 5.8), and 100 mg/L acetosyringone.

[0137] Selection medium; inorganic salts and vitamins of MS medium, 30g/l sucrose, 0.5 mg/l indolebutyric acid, 0.22 mg/l thidiazuron, 8 g/lagar, 5 mM MES (pH 5.8), 25 mg/l G418, and 300 mg/l cefotaxime

Example 7

[0138] Selection of Carnation Transformant

[0139]Agrobacterium tumefaciens LBA 4404 strains each containing pKT11and pKT74 were transformed in accordance with the method of Example 6,and using the obtained G418 resistance seedlings, plants wereregenerated in MS medium containing no plant growth regulatingsubstances (indolebutyric acid, thidiazuron) but G418.

[0140] From the grown plants, plants containing an NPTII gene as aforeign gene were detected by conducting PCR, and it was confirmed thatthe thus-obtained redifferentiated plants were transformants. Here as aprimer for specific amplification of an NPTII gene-specific sequence,the sequences of TAAAGCACGAGGAAGCGGT (SEQ ID NO:5) andGCACAACAGACAATCGGCT (SEQ ID NO:6) were used. The reaction conditiona forPCR were heating at 94° C. for 5 minutes; 30 cycles of 30 seconds at 94°C., 1 minute at 55° C., and 1 minute at 72° C.; followed by a reactionat 72° C. for 10 minutes. In this reaction, ExTaq polymelase(manufactured by Takara Shuzo Co., Ltd.) was used as an enzyme.

[0141] Plants from which the NPTII gene was detected by PCR were used asa transformant. With regard to each of pKT11 and pKT74, thetransformation efficiency ratio was calculated as the number oftransformants per leaf piece used. The ratio for pKT11 was 5% or less,and in contrast the ratio for pKT74 was 25% or more. Incidentally, Gusactivity was determined in the same manner as in Example 3, and it wasconfirmed that all the recombinants expressed the Gus gene.

[0142] In view of the above results, it was considered that MF-48promoter could act on expression of a selection marker gene withremarkable efficiency.

[0143] All publications, patents and patent applications cited hereinare incorporated by reference in their entirety.

1 24 1 320 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA comprising promotor sequence 1 aagcttaaaaggaaggtggc tcctacaaat gccatcattg tgataaagga aaggctatca 60 ttgaagatgcctctacctat agtggtccca aagatggacc cccacccatg aggagcatgg 120 tagaaaaagaagatgttcca accatgtctt caaagcaagt ggattgatgt ggatcctcca 180 atgatgtcaaggatgatgtc aaatcccact atccttgcca agatcttccc tctatataag 240 gaagttcatttcatttggag aggacaaggt actctagact tctttcctca accttctttc 300 ttcttatatatcataccatg 320 2 320 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA comprising promotor sequence 2 aagcttaaaaggaaggtggc tcctacaaat gccatcattg tgataaagga aaggctatca 60 ttgaagatgcctctacctat agtggtccca aagatggacc cccacccatg aggagcatgg 120 tagaaaaagaagatgttcca accatgtctt caaagcaagt ggattgatgt ggatcctcca 180 atgacgtcaaggatgacgtc aaatcccact atccttgcca agatcttccc tctatataag 240 gaagttcatttcatttggag aggacaaggt actctagact tctttcctca accttctttc 300 ttcttatatatcataccatg 320 3 370 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA comprising promotor sequence 3 aagcttaaaaggaaggtggc tcctacaaat gccatcattg tgataaagga aaggctatca 60 ttgaagatgcctctacctat agtggtccca aagatggacc cccacccatg aggagcatgg 120 tagaaaaagaagatgttcca accatgtctt caaagcaagt ggattgatgt ggatcctcca 180 atgatgtcaaggatgatgtc aaatcccact atccttgcca agatcctcca atgatgtcaa 240 ggatgatgtcaaatcccact atccttgcca agatcttccc tctatataag gaagttcatt 300 tcatttggagaggacaaggt actctagact tctttcctca accttctttc ttcttatata 360 tcataccatg370 4 470 DNA Artificial Sequence Description of ArtificialSequenceSynthetic DNA comprising promotor sequence 4 aagcttaaaaggaaggtggc tcctacaaat gccatcattg tgataaagga aaggctatca 60 ttgaagatgcctctacctat agtggtccca aagatggacc cccacccatg aggagcatgg 120 tagaaaaagaagatgttcca accatgtctt caaagcaagt ggattgatgt ggatcctcca 180 atgatgtcaaggatgatgtc aaatcccact atccttgcca agatcctcca atgatgtcaa 240 ggatgatgtcaaatcccact atccttgcca agatcctcca atgatgtcaa ggatgatgtc 300 aaatcccactatccttgcca agatcctcca atgatgtcaa ggatgatgtc aaatcccact 360 atccttgccaagatcttccc tctatataag gaagttcatt tcatttggag aggacaaggt 420 actctagacttctttcctca accttctttc ttcttatata tcataccatg 470 5 19 DNA ArtificialSequence Description of Artificial SequencePrimer 5 taaagcacga ggaagcggt19 6 19 DNA Artificial Sequence Description of Artificial SequencePrimer6 gcacaacaga caatcggct 19 7 42 DNA Artificial Sequence Description ofArtificial SequencePrimer 7 gaatgttaat ttatagatgg ttagagaggt ttatgtagtagg 42 8 26 DNA Artificial Sequence Description of ArtificialSequencePrimer 8 ccatattctc tccaaataaa ataaac 26 9 39 DNA ArtificialSequence Description of Artificial SequencePrimer 9 agtaataattttaggaaatt aaatattttt ttaagaagg 39 10 26 DNA Artificial SequenceDescription of Artificial SequencePrimer 10 tattctctcc aaataaaata aacttc26 11 30 DNA Artificial Sequence Description of ArtificialSequencePrimer 11 ctattccaat ataaacaatt caaaacttac 30 12 37 DNAArtificial Sequence Description of Artificial SequencePrimer 12tgaaatgaat ttttttatat agaggaaggg ttttgtg 37 13 31 DNA ArtificialSequence Description of Artificial SequencePrimer 13 caacataataaaacacaaca cacttatcta c 31 14 36 DNA Artificial Sequence Description ofArtificial SequencePrimer 14 atgaattttt ttatatagag gaagggtttt gtgaag 3615 36 DNA Artificial Sequence Description of Artificial SequencePrimer15 gaagaaattt ttgttaatat ggtggagtat gatatg 36 16 43 DNA ArtificialSequence Description of Artificial SequencePrimer 16 ccaatcaacaaacacataat tacaatctta cacaacatac atc 43 17 34 DNA Artificial SequenceDescription of Artificial SequencePrimer 17 gggatgatgt ataattttattattttttgt aaga 34 18 40 DNA Artificial Sequence Description ofArtificial SequencePrimer 18 cataacatca acttcaaata acatataacc accctaatac40 19 33 DNA Artificial Sequence Description of ArtificialSequencePrimer 19 cttcaataac aaattcattt taacaatcat acc 33 20 31 DNAArtificial Sequence Description of Artificial SequencePrimer 20acaaattcat tttaacaatc ataccttaac t 31 21 42 DNA Artificial SequenceDescription of Artificial SequencePrimer 21 gaggatttaa aaggaaggtggtttttataa atgttattat tg 42 22 38 DNA Artificial Sequence Description ofArtificial SequencePrimer 22 ccacaatttt cacaatccaa actaaatacc cacaaacc38 23 42 DNA Artificial Sequence Description of ArtificialSequencePrimer 23 ggatttaaaa ggaaggtggt ttttataaat gttattattg tg 42 2438 DNA Artificial Sequence Description of Artificial SequencePrimer 24caattttcac aatccaaact aaatacccac aaaccatc 38

1. A DNA represented by the following (a) or (b): (a) a DNA comprising anucleotide sequence consisting of the nucleotide sequence shown in SEQID NO:1 from nucleotide No. 7 to nucleotide No. 272; or (b) a DNAcomprising a nucleotide sequence consisting of the nucleotide sequenceshown in SEQ ID NO:1 from nucleotide No. 7 to nucleotide No. 272,wherein one to several nucleotides are deleted, added or inserted at asite other than nucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78,80 to 82, 109 to 110, 119 to 120, 134 to 135, 145 to 146, 181 to 183,185 to 186, 197 to 198 and 217 to 218, and the deleted, added orinserted sequence is free from any consecutive sequences represented byCG, CAG, CTG, CCG or CGG, and the DNA having promoter activity.
 2. A DNArepresented by the following (c) or (d): (c) a DNA comprising anucleotide sequence consisting of the nucleotide sequence shown in SEQID NO:1; or (d) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:1, wherein one to severalnucleotides are deleted, added or inserted at a site other thannucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to110, 119 to 120, 134 to 135, 145 to 146, 181 to 183, 185 to 186, 197 to198 and 217 to 218, and the deleted, added or inserted sequence is freefrom any consecutive sequences represented by CG, CAG, CTG, CCG or CGG,and the DNA having promoter activity.
 3. A DNA represented by thefollowing (e) or (f): (e) a DNA comprising a nucleotide sequenceconsisting of the nucleotide sequence shown in SEQ ID NO:2 fromnucleotide No. 7 to nucleotide No. 272; or (f) a DNA comprising anucleotide sequence consisting of the nucleotide sequence shown in SEQID NO:2 from nucleotide No. 7 to nucleotide No. 272, wherein one toseveral nucleotide are deleted, added or inserted at a site other thannucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to110, 119 to 120, 134 to 135, 145 to 146, 181 to 183, 183 to 188, 195 to200 and 217 to 218, and the deleted, added or inserted sequence is freefrom any consecutive sequences represented by CG, CAG, CTG, CCG or CGG(where nucleotide nos. 185 to 186 and 197 to 198 are each CG), and theDNA having promoter activity.
 4. A DNA represented by the following (g)or (h): (g) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:2; or (h) a DNA comprising anucleotide sequence consisting of the nucleotide sequence shown in SEQID NO:2, wherein one to several nucleotides are deleted, added andinserted at a site other than nucleotide nos. 41 to 42, 59 to 60, 73 to75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to 135, 145 to 146,181 to 183, 183 to 188, 195 to 200 and 217 to 218, and the deleted,added or inserted sequence is free from any consecutive sequencesrepresented by CG, CAG, CTG, CCG or CGG (where nucleotide nos. 185 to186 and 197 to 198 are each CG), and the DNA having promoter activity.5. A DNA represented by the following (i) or (j): (i) a DNA comprising anucleotide sequence consisting of the nucleotide sequence shown in SEQID NO:3 from nucleotide No. 7 to nucleotide No. 322; or (j) a DNAcomprising a nucleotide sequence consisting of the nucleotide sequenceshown in SEQ ID NO:3 from nucleotide No. 7 to nucleotide No. 322,wherein one to several nucleotides are deleted, added or inserted at asite other than nucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78,80 to 82, 109 to 110, 119 to 120, 134 to 135, 145 to 146, 181 to 183,185 to 186, 197 to 198, 217 to 218, 231 to 233, 235 to 236, 247 to 248and 267 to 268, and the deleted, added or inserted sequence is free fromany consecutive sequences represented by CG, CAG, CTG, CCG or CGG, andhaving promoter activity.
 6. A DNA represented by the following (k) or(l): (k) a DNA comprising a nucleotide sequence consisting of thenucleotide sequence shown in SEQ ID NO:3; or (l) a DNA comprising anucleotide sequence consisting of the nucleotide sequence shown in SEQID NO:3, wherein one to several nucleotides are deleted, added orinserted at a site other than nucleotide nos. 41 to 42, 59 to 60, 73 to75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to 135, 145 to 146,181 to 183, 185 to 186, 197 to 198, 217 to 218, 231 to 233, 235 to 236,247 to 248 and 267 to 268, and the deleted, added or inserted sequenceis free from any consecutive sequences represented by CG, CAG, CTG, CCGor CGG, and the DNA having promoter activity.
 7. A DNA represented bythe following (m) or (n): (m) a DNA comprising a nucleotide sequenceconsisting of the nucleotide sequence shown in SEQ ID NO:4 fromnucleotide No. 7 to nucleotide No. 422; or (n) a DNA comprising anucleotide sequence consisting of the nucleotide sequence shown in SEQID NO:4 from nucleotide No. 7 to nucleotide No. 422, wherein one toseveral nucleotides are deleted, added or inserted at a site other thannucleotide nos. 41 to 42, 59 to 60, 73 to 75, 77 to 78, 80 to 82, 109 to110, 119 to 120, 134 to 135, 145 to 146, 181 to 183, 185 to 186, 197 to198, 217 to 218, 231 to 233, 235 to 236, 247 to 248, 267 to 268, 281 to283, 285 to 286, 297 to 298, 317 to 318, 331 to 333, 335 to 336, 347 to348 and 367 to 368, and the deleted, added or inserted sequence is freefrom any consecutive sequences represented by CG, CAG, CTG, CCG or CGG,and the DNA having promoter activity.
 8. A DNA represented by thefollowing (o) or (p): (o) a DNA comprising a nucleotide sequenceconsisting of the nucleotide sequence shown in SEQ ID NO:4; or (p) a DNAcomprising a nucleotide sequence consisting of the nucleotide sequenceshown in SEQ ID NO:4, wherein one to several nucleotides are deleted,added and inserted at a site other than nucleotide nos. 41 to 42, 59 to60, 73 to 75, 77 to 78, 80 to 82, 109 to 110, 119 to 120, 134 to 135,145 to 146, 181 to 183, 185 to 186, 197 to 198, 217 to 218, 231 to 233,235 to 236, 247 to 248, 267 to 268, 281 to 283, 285 to 286, 297 to 298,317 to 318, 331 to 333, 335 to 336, 347 to 348 and 367 to 368, and thedeleted, added or inserted sequence is free from any consecutivesequences represented by CG, CAG, CTG, CCG or CGG, and the DNA havingpromoter activity.
 9. A DNA strand comprising the DNA according to anyone of claims 1 to
 8. 10. The DNA strand according to claim 9,comprising a structural gene DNA and the DNA of any one of claims 1 to 8which is incorporated at 5′ site of the structural gene DNA in a mannersuch that it is expressed.
 11. A host which is transformed by the DNAstrand of claim 9 or
 10. 12. The host according to claim 11, wherein thehost is a plant cell.
 13. A method for expressing a structural gene in aplant, wherein the host of claim 11 is cultured or cultivated so as toenable the structural gene to be expressed.
 14. The method according toclaim 13, wherein the structural gene is a foreign gene.
 15. A methodfor producing a protein comprising the step of producing the proteinwhich is an expression product of a structural gene whose transcriptionis activated or expression is promoted by a DNA having promoteractivity, using the host of claim
 11. 16. A transformed plant which isobtained by regeneration from the host of claim
 12. 17. The DNA strandaccording to claim 9, comprising a selection marker gene DNA and the DNAof any one of claims 1 to 8 which is incorporated at the 5′ site of theselection marker gene DNA in a manner such that a selection marker geneis expressed.
 18. A method for selecting a transformant host, whereinthe method comprises the steps of: transforming a host with the DNAstrand of claim 17; and culturing the obtained host under a condition inwhich the selection marker gene can be expressed and it can bedetermined whether or not the host expresses the selection marker gene.19. The method according to claim 18, wherein the host is a plant cell.